Ribonucleotide Reductase (RNR) plays an essential role in DNA biosynthesis catalyzing the conversion of nucleotides to deoxynucleotides. Class I and II RNRs are stochiometrically inactivated by ditriphosphates of gemcitabine (F2Cyt) and vinylfluorocytidine (VFCyt). In the Class I enzymes, partial loss of the essential tyrosyl radical is accompanied by the formation of a new nucleotide based radical. Identification of the new substrate radical by CW-EPR spectroscopy was previously complicated by the spectral overlap of the stable tyrosyl radical with the new unidentified radical, which necessitated the use of ambiguous analysis techniques like spectral subtraction. Recently, we reinvestigated the EPR signal obtained after incubation of Class I RNR from E. coli with 2'-azido-2'-deoxyuridine 5'-diphosphate (N3UDP) and utilized high sensitivity pulsed EPR spectroscopy at 140 GHz. Electron-spin-echo detected spectra at 10 K were achieved with excellent signal-to-noise (> 100). Pulsed EPR detection allows separation of radical species if those are characterized by substantially different T, spin-lattice relaxation times. We found that T, of the tyrosyl radical amounts to about 8 ms at 10 K and is considerably shorter than the relaxation rate of typical isolated organic radicals (TI > 50 ms). We attribute the short relaxation time to a strong exchange interaction with the essential diiron cluster in the B2 subunit. The relaxation rate strongly increases with temperature and at about 80 K the spin-echo signal of the tyrosyl radical disappears during the short stimulated echo sequence. We demonstrated that at 80 K the EPR spectrum recorded after enzyme inhibition only consists of the new unknown nucleotide radical and that an unambiguous simulation of the hyperfine and g-tensors was feasible. Application of pulsed EPR to study the unknown mechanism of inhibition of RNR with gemcitabine is currently under investigation.